title: Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum.
abstract: Fusarium oxysporum is a devastating plant pathogen that oxidizes C₁₈ fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C₁₈ and C₂₀ fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu⁹⁴⁶-Val-Leu-Ser⁹⁴⁹, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, SE-75124 Uppsala, Sweden.
authors.forename: Inga
authors.initials: I
authors.lastname: Hoffmann
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D019295:Computational Biology; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017962:alpha-Linolenic Acid / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D017962:alpha-Linolenic Acid; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase
keywords: cytochrome P450 class III; heme peroxidase; linoleate diol synthase; mutagenesis site-specific; oxygenation mechanism; oxylipin biosynthesis